I am used to work with BT474 cell line. But as I was needing a huge amount of cells, I expanded them in 4 flask T150 during 3 weeks. It is known that the growth is very slow, but suddenly, when the confluence reaches about 70%, the growth speed increases. So in order to take care the cells before use them, I split them half-half, I froze one part and kept other in culture. On the next day, about 80% of the cells were detached into the flask, looking like dead. I tried to take care, changing the medium again, but it did not work, unfortunately.
Please, someone knows if is not good to got the maximum confluence in this cell? Or knows what it happened?
Thank you!
I have used BT-474 briefly in the past, but did not culture them long term or in large quantities. I recall them growing slowly, but not having much issue passaging them. If I recall correctly, I would culture them in DMEM w/ 10% FBS and Pen/Strep and passage them 1:3 or 1:4 when they reach 80% confluence. I have attached a link to the recommended culture methods for BT-474 from ATCC. They suggest another media, but the only other additive is 10% FBS.
Perhaps the cells arrest upon reaching high confluence and are no longer recoverable.
http://www.atcc.org/products/all/HTB-20.aspx#culturemethod
Dear Priscila,
BT-474 are groowing very slowly. Do not wait/ expect 100 % confluency, maximum they reach is about 80%. I think you can try with DMEM medium +20% FCS but rather I would try with medium they recommend on atcc:
http://www.lgcstandards-atcc.org/products/all/HTB-20.aspx?geo_country=ch#culturemethod
there you can also find more about the behavior of the cells and culture conditions.
I grow these using DMEM and 10% FCS + Pen Strep. Split when 70% confluent. To grow in bulk, you need to use more flasks!!!
Dear Priscila,
I've cultured BT474. I've used DMEM/F12+10%FBS and p150 plates (but of course you can use flasks). I would recommend you to grow them until 80% confluence and split them 1:2 or 1:3 but not more than that, as these cells are really sensitive to being diluted and the growth will be impaired. I typically split them 1:2. I only do 1:3 when they get around 90% confluent. I hope it helps. Good luck!
Mariano
Maybe I kept these cells so long in culture, and as I tried to split them, they were sensitive and died. I have new BT474 cells in culture now, and it looks like good!
Thank you, everybody!
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